Evaluation of endoscopic vein extraction on structural and functional viability of saphenous vein endothelium

Abstract

Objectives: Endothelial injury during harvest influences graft patency post CABG. We have previously shown that endoscopic harvest causes structural and functional damage to the saphenous vein (SV) endothelium. However, causes of such injury may depend on the extraction technique. In order to assess this supposition, we evaluated the effect of VirtuoSaph endoscopic SV harvesting technique (VsEVH) on structural and functional viability of SV endothelium using multiphoton imaging, biochemical and immunofluorescence assays.

Methods: Nineteen patients scheduled for CABG were prospectively identified. Each underwent VsEVH for one portion and "No-touch" open SV harvesting (OSVH) for another portion of the SV. A two cm segment from each portion was immersed in GALA conduit preservation solution and transported overnight to our lab for processing. The segments were labeled with fluorescent markers to quantify cell viability, calcium mobilization and generation of nitric oxide. Morphology, expression, localization and stability of endothelial caveolin, eNOS, von Willebrand factor and cadherin were evaluated using immunofluorescence, Western blot and multiphoton microscopy (MPM).

Results: Morphological, biochemical and immunofluorescence parameters of viability, structure and function were well preserved in VsEVH group as in OSVH group. However, tonic eNOS activity, agonist-dependent calcium mobilization and nitric oxide production were partially attenuated in the VsEVH group.

Conclusions: This study indicates that VirtuoSaph endoscopic SV harvesting technique preserves the structural and functional viability of SV endothelium, but may differentially attenuate the vasomotor function of the saphenous vein graft.

Figure 1

Multiphoton images of SV in the transmission mode. Endothelium and smooth muscle cells do not show visible damage and remain intact in vessels harvested by both techniques. OSVH: open saphenous vein harvest; VsEVH: VirtuoSaph endoscopic harvest. Magnification 400X Figure 1a. Intact saphenous vein Figure 1b. Frozen sections: 40 μ m

Figure 2

Esterase activity in the OSVH and VsEVH samples: Representative images showing similar esterase activity and viability (green fluorescence) in both samples (a and b). Both techniques caused minimal visible damage to the vessels as indicated by attenuated red fluorescence in endothelial and smooth vessel regions (c and d). Magnification = 400 ×.

Figure 3

Bradykinin mediated calcium mobilization and NO production in SV; (A) Representative images of calcium mobilization pre and post bradykinin stimulation in the OSVH and VsEVH groups. (B) Representative images of nitric oxide production pre and post bradykinin stimulation. Magnification = 400 x.

Figure 4

Quantitative representation of normalized calcium mobilization and nitric oxide production in the OSVH and VsEVH groups. Bradykinin induced calcium mobilization and nitric oxide production was greater in the OSVH group over baseline than in the VsEVH group. N = 95 measurements for each group.

Figure 5

Immunofluorescent labeling of endothelial cell markers in SV samples. Representative fluorescence images showing normal distribution of caveolin, eNOS, von Willebrand factor and cadherin in both, OSVH and VsEVH groups. Harvesting of SV using the VirtuoSaph endoscopic technique did not damage or alter the localization of proteins in the endothelium. Magnification = 400 x.

Figure 6

Western blot analysis of SV harvested by OSVH and VsEVH technique demonstrates similar resolution and concentration of endothelial proteins. Endoscopic harvesting using VirtuoSaph did not negatively affect the endothelial proteins in SV samples. Samples 1-6: OSVH Group; Corresponding samples 7-12: VsEVH Group.